1. Field of the Invention
The present invention relates to an improved method and apparatus for carrying out solid phase in vitro diagnostic assays.
2. Description of the Prior Art
In recent years, numerous techniques have been employed in the area of laboratory diagnostics to simplify operating procedures of existing methods and to provide new methods of improved, speed, sensitivity, and accuracy. In particular, solid phase reactions have been especially valuable in simplifying the manipulations of prior art procedures and making possible procedures that could not be performed with conventional homogeneous phase reactions.
A solid phase reaction is generally carried out between one reactant, the fixed component, immobilized on the surface of an insoluble support matrix, and a second reactant, the mobile component, in solution. The reaction occurs when a molecule or a molecular arrangement of the mobile reactant, in the course of diffusion, collides with a molecule of the fixed reactant immobilized on the surface of the solid support matrix. The reaction may be a conventional chemical reaction, a binding of the mobile component by the fixed component as in an immunochemical reaction between an antigen and an antibody, or it may be a binding of the mobile component by the fixed component accompanied by chemical transformation of one of the components such as occurs in an enzyme-catalyzed reaction. Quantitative results are obtained by measuring the formation of products or disappearance of reactants as in the case of conventional and enzyme-catalyzed reactions, and in measuring the amount of the mobile component bound or the amount of mobile component unbound, in the case of an immunochemical reaction.
Any conventional chemical reaction or enzyme-catalyzed reaction resulting in a directly or indirectly measurable change can, in principle, be carried out by solid phase techniques. Directly measurable changes include changes in pH, light absorbance in the visible and ultraviolet regions or changes in fluorescence intensity. Indirect measurements can be made whenever the primary reactants or products are not readily measurable themselves by interposing the action of a reagent to carry out further reaction steps resulting in a measurable change and by the introduction of specific separation techniques. Such strategies may be employed alone or in combination, as is understood in the art.
Where the reaction consists solely of binding, in the absence of chemical change, techniques developed in the field of immunochemistry may be used to measure the extent of the reaction. Solid phase reactions are especially suited for immunochemical assays because the reactants in bound form may readily be removed from the solution by virtue of their attachment to the solid phase. Frequently, however, the components bound in an immunochemical reaction cannot be directly measured because they are indistinguishable by chemical methods from other substances commonly present in the same reaction mixture, so that the mere disappearance of a reactive component from solution or its accumulation on the solid phase cannot be measured directly. Therefore, additional steps must be taken in order to make a measurable change related to the amount of binding.
The variety of approaches taken by workers in the prior art can be grouped into two general categories. In the first of these, termed competitive or indirect immunoassays, the immobilized component is present in controlled amount and the mobile component present in unknown amount. To the unknown amount of mobile component is added a known amount of the same component which has been tagged by the addition of a measurable substituent which does not interfere with its immunochemical reactive properties. The tag may consist of a radioisotope, a chromophore, a fluorophor or an enzyme. The amount of tagged material bound immuno-chemically to the solid phase will depend upon the amount of untagged component in solution competing for the same binding sites. The more of the unknown present, the less will be the amount of tagged component bound.
In the second general method, termed the sandwich method or direct method, the solid phase containing an amount of immunochemically bound mobile component resulting from the first immunochemical reaction is subjected to the action of a reagent which can also bind immunochemically to the solid phase, but only at sites already occupied by the immunochemically bound mobile component. The reagent may be tagged, for example, as in the first method with a radioisotope, a fluorophor, a chromophore or an enzyme. The amount of tagged reagent bound is a direct meaasure of the amount of mobile component bound, which, in turn, is a measure of the amount of mobile component initially present in the reaction mixture.
Where the tag is a radioisotope, the technique, whether competitive or noncompetitive, is termed a radioimmunoassay. When the tag is an enzyme, the assay is termed an enzyme-linked immunoassay. The amount of enzyme-tagged reactant is measured by any convenient method for measuring the activity of the enzyme used in the tag.
Other kinds of solid phase reactions of the type generally described hereinabove are presented by way of example. The immunoradiometric assay for quantitative determination of an antigen is conducted by first reacting a known excess of labeled antibody with the unknown amount of antigen in a homogeneous phase reaction. Subsequently, immobilized antigen in excess amount is added in order to bind the unreacted soluble labeled antibody. The amount of unknown antigen is determined by measuring the difference between the total labeled antibody and the amount bound to the solid phase. The method gives direct quantitative results only with an univalent antigen, i.e., antigen which can only bind one molecule of antibody.
Immunochemical assays are highly useful in clinical research and diagnosis. They are highly specific, owing to the highly selective nature of antigen-antibody reactions. The antigen-antibody binding is very tight so that once the binding reaction has had an opportunity to occur, the limit of detectability is determined by the measurability with which the tag can be detected. Immunochemical assays are exceedingly versatile, owing to the fact that they can be used to measure specific substances selectively against a background of chemically similar substances. Because of these desirable attributes, there has been considerable interest in improving the ease of manipulation, sensitivity, accuracy, speed and applicability of immunochemical assays. The development of solid phase immunoassays has been one of the major advances in the field.
Among the advantages of solid phase systems is that the reaction product or products can be separated from the reaction solution with relative ease, i.e., by physically removing the solid phase material. This is in contrast with a non-solid phase or a homogeneous reaction, which typically results in a homogeneous solution which requires more complex separation techniques.
The introduction of solid phase technology has permitted the performance of novel procedures that were heretofore extremely difficult using free solution technology. An example of this is the sandwich assay technique described hereinabove. While a sandwich assay is theoretically possible in a homogeneous solution, it is not desirable for practical reasons. The most important aspect which makes such assays impractical is that the separation of the first antigen-antibody complex from a homogeneous phase solution requires the use of sophisticated physical-chemical techniques, especially if the antigen is relatively small compared to the antibody and molecular weight differences between free antibody and complexed antibody are slight. The separation procedure in a solid phase system, by contrast, is a matter of relative simplicity.
In solid phase technology, the reagent or reagents used in the procedure are usually immobilized by being coated or bonded, either covalently or by adsorption to the solid phase material, which is then immersed in the sample to be tested. The manner of coupling such reagents to the solid phase material is known. See, for example, the disclosures in U.S. Pat. No. 3,652,761, U.S. Pat. No. 3,879,262 and U.S. Pat. No. 3,896,217.
Examples of commonly used solid phase materials include, but are not limited to, glass or polymeric tubes which are coated with the reagent or reagents on their internal surfaces, coated polymeric inserts, micro and macro beads formed of polymers and of glass, porous matrices, coated membranes, and tablets.
Particularly useful are the coated polymeric insert matrices described in copending U.S. application Ser. No. 905,552, of Piasio et al., filed May 15, 1978 and assigned to the assignee of the present invention. The coated inserts described herein include a handle member having attached at one end a plurality of elements having essentially smooth plane or curved surfaces with the fixed component immobilized thereon. These inserts, which in the preferred embodiment take the form of a central rod having a number of fins extending outwardly from the rod along a portion of its length, are characterized by a large ratio of solid phase surface area to fluid sample volume and by a short average diffusion distance between the molecules of mobile component in the fluid sample and the fixed component distributed on the solid phase surface. These factors contribute to an enhancement of the reaction rate and a consequent reduction in the time required to carry out a given diagnostic assay.
Moreover, since the inserts described in copending application Ser. No. 905,552 extend substantially throughout the depth of the fluid sample and preferably above the surface of the fluid sample as well, an essentially constant geometric relationship exists between the fluid volume and the solid phase surfaces of the insert matrix throughout the depth of the fluid sample, such relationship being preserved despite changes in the fluid volume. This factor tends to produce more uniform results and to minimize the effect of human error when a number of assays are performed sequentially or concurrently.
As disclosed in copending U.S. application Ser. No. 064,389, of Piasio et al., filed Aug. 8, 1979 and also assigned to the assignee of the present invention, the reaction kinetics may be further enhanced by coating the inner surface of the fluid receptacle with the same fixed component that is applied to the surfaces of the solid phase insert matrix. This has the effect of further increasing the ratio of the solid phase surface area to fluid sample volume and hence the effective concentration of the fixed component, and further reducing the average diffusion distance between the mobile and fixed components. In this way reaction equilibrium is reached more quickly, and the reliability of the assay improved, as compared with assays conducted using coated inserts or coated receptacles alone.
Despite the high level of refinement exhibited by present-day solid phase immunoassay techniques, certain difficulties still exist when immunoassays are performed on a large scale, as it typically the case in hospitals and clinical testing laboratories. These difficulties arise not from any inherent limitations in the basic solid phase reaction technology, but rather from the redundant and time-consuming physical manipulations that are required for carrying out multiple simultaneous or sequential immunoassays using presently available equipment.
The nature of the above-mentioned manipulative difficulties may be readily apprehended by first considering a competitive or indirect radioimmunoassay carried out using a coated polymeric insert and a fluid receptacle (e.g., a test tube) for receiving the insert and the fluid sample to be assayed. The insert may take the form of a central rod or stick having a number of fins extending outwardly from the rod along a portion thereof, as described in the aforementioned copending application Ser. No. 905,552. The fins may, but need not, be made to conform approximately to the shape of the test tube or other receptacle in which the insert will be received. The portion of the central rod not having fins attached thereon serves as a handle member, facilitating the introduction of the fin-bearing portion of the insert into the fluid receptacle and avoiding the necessity of touching and possibly contaminating the antibody immobilized on the fin surfaces.
Typically, the fluid sample containing both the unknown and radioactively tagged mobile components is introduced into the test tube prior to the insertion of the finned insert matrix into the tube. The subsequent introduction of the insert matrix into the test tube then marks the start of the reaction for timing purposes. Alternatively, the fluid sample may be introduced into the test tube with the insert matrix already in place therein, which will be the procedure followed when, for example, the fixed component is immobilized on the inside surface of the test tube as well as on the reactive surfaces of the insert matrix, as taught in the aforementioned copending application Ser. No. 064,389. The finned stick is a particularly suitable insert when this alternative procedure is elected, since the open interstices between adjacent fins permit substantially unimpeded pouring of the fluid sample into the receptacle while the insert is in place therein.
After a suitable reaction interval has elapsed, the reaction is stopped by separating the insert matrix from the fluid sample so that no further binding of the mobile components to the fixed component can take place. This is usually done by removing the insert matrix from the reaction tube and washing it in a further test tube containing water or a wash buffer. The washing operation removes most of the tagged and untagged mobile components that are not actually immunochemically bound to the antibody immobilized on the fin surfaces of the insert matrix. After washing, the insert matrix is removed from the wash fluid and placed in a further clean test tube for measurement in a suitable radioactive counting chamber. A scintillation fluid is often introduced into the test tube used in this measurement in order to enhance the radioactive count obtained.
It is apparent that the procedure outlined above requires at least three test tubes and two insert matrix removal and reinsertion operations in order to carry out a single complete assay. It is possible to reduce the number of test tubes required to two by rinsing either the reaction tube or the wash tube and re-using it for the measuring operation, but this increases the number of manipulative operations that the technician must perform and hence the time required for the assay as a whole. The use of fewer than two tubes would not be in accord with proper laboratory procedure, since a second test tube is required for safely confining the now-radioactive insert while the first is decanted and rinsed for re-use in the measuring operation. The insert might easily slip out of its test tube if decanting and rinsing were to be attempted while it was in place, possibly contaminating the laboratory environment or the reactive surfaces of the insert as a result.
In the case of the sandwich or direct radioimmunoassay method, it will be recalled that two distinct immunochemical reactions must be carried out. The first is the binding of the unknown mobile component to be fixed component immobilized on the solid phase insert surfaces, and the second is the binding of the tagged reagent to the sites on the solid phase matrix already occupied by the unknown mobile component that was bound during the first reaction. Since these two reactions are usually carried out sequentially, using separate reaction fluids for the respective unknown and tagged mobile components, it is apparent that the direct radioimmunoassay will ordinarily require one more test tube than the indirect radioimmunoassay described previously. While it is possible to carry out the two reactions essentially simultaneously in the same tube, it is clear that the number of test tubes and manipulative operations required to perform the assay will still be at least as great as in the case of the indirect or competitive method.
Viewed in the context of a single assay, the number of test tubes and manipulative operations required may not appear to be a major consideration. In hospitals and testing laboratories where assays are regularly performed in large numbers, however, these factors take on considerable importance. The number of clean test tubes that must be kept on hand to carry out a particular assay method, and the time required for performing the various manual operations attending its use, may well be determinative of the commercial feasibility of adopting the assay method for large scale use.
From the standpoint of laboratory procedure, prior art coated tube assays possessed the undeniable advantage of simplicity, requiring no assembly or disassembly steps and usually no additional test tubes for performing the assay. The improved reaction kinetics made possible by the advent of the coated insert matrix, however, render these assays more desirable than coated tube methods despite the added procedural complexity involved in handling the insert as a separate and removable part of the assay assembly. Ideally, it would be desirable to combine the procedural advantages stemming from unitary structure, as exhibited by the coated tube assay systems, with the flexibility and reaction efficiency that characterize coated insert systems.
This ideal has been realized only imperfectly in the prior art. U.S. Pat. No. 4,116,638, for example, describes a solid phase support structure consisting of a number of capillary tubes retained by a circular disk which forms an airtight seal against the interior of a reaction vessel. A central tube of greater diameter than the capillary tubes is said to be usable for the introduction of reactive and wash solutions into the vessel or for their removal from the vessel by suction, but the introduction of a fluid into the vessel by a simple manual pouring operation would appear to be impractical given the narrow diameter of the central tube relative to the diameter of the mouth of the vessel. Moreover, it would appear that the circular disk would prevent complete decanting of the fluid in the vessel if the whole apparatus were simply inverted. In order to insure complete draining of the vessel, therefore, the technician performing the assay must either attach an external source of suction to the central tube, or remove the support structure as a whole from the reaction vessel.
U.S. Pat. No. 4,066,646 describes a combined diagnostic device and tubular housing assembly which includes a cap for sealing the open end of the tubular housing. The cap also serves as the support for a depending rod which extends downwardly toward the fluid sample contained in the tubular housing. The lower end of the rod in turn supports a thin sheet of substrate material having a biologically active layer thereon for immersion in the fluid sample. Although an essentially unitary structure is thereby provided, it is apparent that the cap prevents pouring of a fluid sample into the tubular housing, as well as decanting of the fluid sample and rinsing of the substrate, while the apparatus remains assembled.